Design and fabrication of hydrogel scaffolds for osteochondral tissue regeneration

Abstract

Osteochondral defects are serious clinical problems relating to damaged articular cartilage within joints, usually resulting from acute traumatic injury or an underlying bone disorder. A variety of therapeutic options have been investigated, with several commercial products addressing this problem, but with limitations in the technology used in terms of biomaterials and construct presentation. Many of the current devices used to ‘plug’ the osteochondral defect presents a laminated structure which will interact with bone and cartilage layers within the damaged site. These devices do not offer reconstruction of normal tissue architecture and may delaminate in worse cases resulting in pain and repeated surgical intervention.
Advances in biomaterial design and tissue engineering offer promise for the development of new approaches to direct cell architecture and tissue formation. The current work focuses on testing the impact of biomaterial chemistry and presentation in 3D, using a poly (N-isopropylacrylamide) (pNIPAM), and slightly less wettable poly (N-tert-butylacrylamide) (pNTBAM) to support spatial control of osteogenic and chondrogenic growth. Both materials were demonstrated as single component hydrogel, and presented in gradient form, in order to steer attachment of these two cell types.
Both materials were prepared using ion transfer radical polymerization. FTIR spectroscopy and water droplet angle measurements used to describe main chemical variations and the wettability profile. Mechanical testing determines materials strength and stiffness, while scanning electron microscopy (SEM) defines architectural and pore differences. Bio-glass (BG) fibres were embedded within hydrogels to support mineral environment and aid in cellular transportation. Histological staining using H&E stain together with confocal imaging used to configure cell attachment upon each hydrogel. Cell survival was examined using live/dead staining of hydrogel samples for immortalized cell lines (MG63, OK3H) and primary cell lines including human osteoblasts (hOBs) and human chondrocytes (hCHs). Osteogenic and chondrogenic potential of cells were investigated with alizarin red staining and calcium assay. Alcian blue and dimethyl methylene blue were used to assess glycosaminoglycan (GAG) production. Protein assessment was performed using immunostaining and ELISA assay for collagens I, and II as a marker for cell function in addition to collagen X and ELISA quantification of annexin A2 as a markers for mineralization.
Results indicated more hydrophobic stiffer mass for pNTBAM compared to pNIPAM. Internal architecture revealed larger pore diameter measured for pNIPAM hydrogel. Viability of all cell types was found to be good on both gel types, although proliferation was higher on pNTBAM compared to pNIPAM, and the latter gave rise to greater number of cell aggregates. Both hydrogels supported mineralization and GAG production, with pNTBAM presenting higher amounts mostly for GAGs. Higher levels of mineralization were obtained with BG embedded samples. These results were confirmed by detecting collagens and annexin A2 levels.
In conclusion, the various characteristics for pNIPAM and pNTBAM impacted the biological observations in terms of survival and cell function. This was useful in establishing a combined multi-regional scaffold which revealed the development of mineral and cell functional gradient between the scaffold’s sides.